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Nucleosomes around the promoter region are disassembled for transcription in response to various signals, such as acetylation and methylation of histones. Although the interactions between histone-acetylation-recognizing bromodomains and factors involved in nucleosome disassembly have been reported, no structural basis connecting histone modifications and nucleosome disassembly has been obtained. Here, we determined at 3.3 [Angstrom] resolution the crystal structure of histone chaperone cell cycle gene 1 (CCG1) interacting factor A/antisilencing function 1 (CIA/ASF1) in complex with the double bromodomain in the CCG1/TAF1/TAF(II)250 subunit of transcription factor lID. Structural, biochemical, and biological studies suggested that interaction between double bromodomain and CIA/ASF1 is required for their colocalization, histone eviction, and pol II entry at active promoter regions. Furthermore, the present crystal structure has characteristics that can connect histone acetylation and CIA/ASFl-mediated histone eviction. These findings suggest that the molecular complex between CIA/ASF1 and the double bromodomain plays a key role in site-specific histone eviction at active promoter regions. The model we propose here is the initial structure-based model of the biological signaling from histone modifications to structural change of the nucleosome (hi-MOST model). chromatin | transcription | transcription factor IID | x-ray crystallography doi/10.1073/pnas.0912509107

Histone chaperones assemble and disassemble nucleosomes in an ATP-independent manner and thus regulate the most fundamental step in the alteration of chromatin structure. The molecular mechanisms underlying histone chaperone activity remain unclear. To gain insights into these mechanisms, we solved the crystal structure of the functional domain of SET/TAF-I[beta]/INHAT at a resolution of 2.3 [Angstrom]. We found that SET/TAF-I[beta]/INHAT formed a dimer that assumed a 'headphone'-like structure. Each subunit of the SET/ TAF-I[beta]/INHAT dimer consisted of an N terminus, a backbone helix, and an 'earmuff' domain. It resembles the structure of the related protein NAP-1. Comparison of the crystal structures of SET/TAF-I[beta]/INHAT and NAP-1 revealed that the two proteins were folded similarly except for an inserted helix. However, their backbone helices were shaped differently, and the relative dispositions of the backbone helix and the earmuff domain between the two proteins differed by [approximately equal to]40[degrees]. Our biochemical analyses of mutants revealed that the region of SET/TAF-I[beta]/INHAT that is engaged in histone chaperone activity is the bottom surface of the earmuff domain, because this surface bound both core histones and double-stranded DNA. This overlap or closeness of the activity surface and the binding surfaces suggests that the specific association among SET/TAF-I[beta]/INHAT, core histones, and double-stranded DNA is requisite for histone chaperone activity. These findings provide insights into the possible mechanisms by which histone chaperones assemble and disassemble nucleosome structures. nucleosome | chromatin | transcription | replication | repair

General transcription initiation factor IID (TFIID) plays a central and critical role in transcription initiation from both naked and chromatin templates. Although interaction between several DNA-binding proteins and TFIID were identified and well characterized, functional linkage between TFIID and chromatin factors has remained to be elucidated. Here we show the identification and characterization of human CIA/hASF1 (identified previously as a histone chaperone) as an interactor of two tandem bromodomain modules of human (h)(TA[F.sub.II])250/CCG1, the largest subunit of TFIID. Although yeast (y)(TA[F.sub.II])145, a homologue of (hTA[F.sub.II])250/CCG1 in Saccharomyces cerevisiae, lacks bromodomains, glutathione S-transferase pull-down and immunoprecipitation assays revealed that Asf1p (antisilencing function 1), the counterpart of CIA in S. cerevisiae, interacts with Bdf1p (bromodomain factor 1), which is reported to serve as the missing bromodomain in (yTA[F.sub.II])145. Furthermore, yeast strain lacking the BDF1 gene shows the Spt phenotype that is shown also by the ASF1 gene disruptant, and a double-knockout strain of both genes shows synthetic lethality, indicating that ASF1 genetically interacts with bromodomains associated with yTFIID. We also found that Asf1p coprecipitates with yTFIID subunits from yeast whole-cell extract, and overexpression of yTFIID subunits suppress the Spt phenotype caused by gene disruption of the ASF1. This study describes the functional linkage between TFIID and a histone chaperone.

The transcription initiation factor TFIID, consisting of the TATA box-binding protein (TBP) and many TBP-associated factors (TAFs), plays a central role in both basal and activated transcription. An intriguing finding is that the 80-residue N-terminal region of Drosophila [TAF.sub.II]230 [[dTAF.sub.II]230-(2-81)] can bind directly to TBP and inhibit its function. Here, studies with mutated forms of TBP demonstrate that [dTAF.sub.II]230-(2-81) binds to the concave surface of TBP, which is important for TATA box binding. Previously, it was reported that a point mutation (L114K) on this concave surface destroys the ability of TBP to bind VP16 and to mediate VP16-dependent activation in vitro, but has no effect on basal transcription. Importantly, the same TBP mutation eliminates TBP binding to [dTAF.sub.II]230-(2-81). Consistent with these effects of the L114K mutation, [dTAF.sub.II]230-(2-81) and the VP16 activation domain compete for binding to wild-type TBP. These results indicate that transcriptional regulation may involve, in part, competitive interactions between transcriptional activators and TAFs on the TBP surface.

An analysis of the human transcription initiation factor, TFIID, composed of the TATA-binding polypeptide (TBP) and TBP-associated factors (TAFs) revealed that TAF(sub II)80 interacts with TAF(sub II)250, TAF(sub II)31, TAF(sub II)20, and TAP while TAF(sub II)80 interacts with TRIIEalpha and TRIIFalpha. The mutation of TAF(sub II)80 indicated three distinct interaction domains falling within regions conserved in human TAF(sub II)80, Drosophila TAF(sub II)60, and yeast TAF(sub II)60. This process of isolating these cDNAs are discussed.

The largest subunit of the transcription initiation factor (TFIID), p230, contained two interaction sites with TATA-box binding polypeptide, with the N-terminal site mediating the inhibition of TBP function and the strong physical interactions with TBP. The multimeric protein, Drosophila TFIID, which is important for transcriptional regulation consists of TBP and several other polypeptides.

Transcription initiation factor IID (TFIID) is composed of a TATA-binding subunit, TBP, and other polypeptides which may be involved for promoter interactions and activator-dependent transcription. Using a TBP-specific antibody column, a number of human TFIID polypeptides which are associated with TBP were isolated. Further characterization of these polypeptides demonstrated that at least one of them could interact directly with TBP. These polypeptides may play a major role in the assembly and function of TFIID.

Results of experiments designed to investigate the domain responsible for transcription factor IID (TFIID) DNA binding show that mutations of conserved residues in the interrupted direct repeat regions of TFIID eliminates its DNA binding property. However, neither mutations in the lysine residues which comprise the intervening basic repeat region nor nucleotide alterations of non-conserved residues, even if located in the directrepeat region, affects DNA binding. These findings suggest that the binding region has a bipartite nature and consists of domains which dictate spatial structure orientation.

The Drosophilia gene encoding transcription factor IIB (dTFIIB) was isolated and characterized using polymerase chain reaction amplification. Results show that the predicted amino acid sequence of the dTFIIB gene product was 79% identical and 85% similar to human and Xenopus TFIIB sequences. TFIIB was also noted to contain structural direct repeats, basic repeats and sigma sequences similar tothe TATA binding subunit of TFIID. Studies also reveal that the N-terminal sequences of TFIIB is highly conserved among most species indicating that it probably plays a role in basal transcription.

Eukaryotic promoters are activated and regulated with the assistance of TFIID a general transcription initiation factor. Unusual structural motifs in the DNA-binding domain typifies the unique binding properties of TFIID and its other roles. TFIID can cause DNA bending on the area of the TATA element and this might start the pooling of other initiation factors into a functional preinitiation mechanism. The observed bending may also possess a stabilizing effect on factor interactions.

Currently, there is no method to distinguish between the roles of a subunit in one multisubunit protein complex from its roles in other complexes in vivo. This is because a mutation in a common subunit will affect all complexes containing that subunit. Here, we describe a unique method to discriminate between the functions of a common subunit in different multisubunit protein complexes. In this method, a common subunit in a multisubunit protein complex is genetically fused to a subunit that is specific to that complex and point mutated. The resulting phenotype(s) identify the specific function(s) of the subunit in that complex only. Histone H2B is a common subunit in nucleosomes containing H2A/H2B or Htz1/H2B dimers. The H2B was fused to H2A or Htz1 and point mutated. This strategy revealed that H2B has common and distinct functions in different nucleosomes. This method could be used to study common subunits in other multisubunit protein complexes. [ABSTRACT FROM AUTHOR]

Histone chaperones assemble and disassemble nucleosomes in an ATP-independent manner and thus regulate the most fundamental step in the alteration of chromatin structure. The molecular mechanisms underlying histone chaperone activity remain unclear. To gain insights into these mechanisms, we solved the crystal structure of the functional domain of SET/TAF-Iβ/INHAT at a resolution of 2. Å. We found that SET/TAF-Iβ/INHAT formed a dimer that assumed a ‘headphone’-Iike structure. Each subunit of the SET/TAF-Iβ/INHAT dimer consisted of an N terminus, a backbone helix, and an ‘earmuff’ domain. It resembles the structure of the related protein NAP-l. Comparison of the crystal structures of SET/TAF-Iβ/INHAT and NAP-1 revealed that the two proteins were folded similarly except for an inserted helix. However, their backbone helices were shaped differently, and the relative dispositions of the backbone helix and the earmuff domain between the two proteins differed by ≈40°. Our biochemical analyses of mutants revealed that the region of SET/TAF-Iβ/INHAT that is engaged in histone chaperone activity is the bottom surface of the earmuff domain, because this surface bound both core histones and double-stranded DNA. This overlap or closeness of the activity surface and the binding surfaces suggests that the specific association among SET/TAF-Iβ/INHAT, core histones, and double-stranded DNA is requisite for histone chaperone activity. These findings provide insights into the possible mechanisms by which histone chaperones assemble and disassemble nudeosome structures. [ABSTRACT FROM AUTHOR]

Histone chaperones assemble and disassemble nucleosomes in an ATP-independent manner and thus regulate the most fundamental step in the alteration of chromatin structure. The molecular mechanisms underlying histone chaperone activity remain unclear. To gain insights into these mechanisms, we solved the crystal structure of the functional domain of SET/TAF-Ibeta/INHAT at a resolution of 2.3 A. We found that SET/TAF-Ibeta/INHAT formed a dimer that assumed a "headphone"-like structure. Each subunit of the SET/TAF-Ibeta/INHAT dimer consisted of an N terminus, a backbone helix, and an "earmuff" domain. It resembles the structure of the related protein NAP-1. Comparison of the crystal structures of SET/TAF-Ibeta/INHAT and NAP-1 revealed that the two proteins were folded similarly except for an inserted helix. However, their backbone helices were shaped differently, and the relative dispositions of the backbone helix and the earmuff domain between the two proteins differed by approximately 40 degrees . Our biochemical analyses of mutants revealed that the region of SET/TAF-Ibeta/INHAT that is engaged in histone chaperone activity is the bottom surface of the earmuff domain, because this surface bound both core histones and double-stranded DNA. This overlap or closeness of the activity surface and the binding surfaces suggests that the specific association among SET/TAF-Ibeta/INHAT, core histones, and double-stranded DNA is requisite for histone chaperone activity. These findings provide insights into the possible mechanisms by which histone chaperones assemble and disassemble nucleosome structures.